Kelly Hypoxia RNA-Seq

RNA-Seq of Hif1a, Hif2a & Hif1b gene knock-outs

1.) Data processing

combine sample list

experiment RNAs conditions date seq_id Seq_runs
3 Katharina 16 Kelly_Nx Kelly_Hx HIF1A_Hx HIF2A_Hx 2018-09-13 2018-09-14 P557 16
1 Simon 22 Kelly_Nx Kelly_Hx HIF1A_Nx HIF1A_Hx HIF1B_Nx HIF1B_Hx 2017-05-04 2021-06-16 2021-08-25 2021-08-27 P2041 22
2 Ulrike 50 Kelly_Nx Kelly_Hx HIF1A_Nx HIF1A_Hx HIF2A_Nx HIF2A_Hx 2023-06-02 2023-06-08 2023-06-15 2023-06-28 P3302 150

DESeq2 Design

design = ~experiment+genotype+treatment+genotype:treatment

MA plot & Dispersion

Transformations

Sample distance

Principal component analysis

Plot example counts

  1. network analysis

  1. Differential gene expression

Contrasts_overview

Contrasts_overview

## cutoffs ## differential expressed: p=0.05,bM=10,l2FC=1 ## top genes: p=0.01,bM=100,l2FC=2

all.DEGs top.DEGs
1
deg_Hif1a.Hx.vs.Nx 6166 983
deg_Hif2a.Hx.vs.Nx 3394 542
deg_Hif1b.Hx.vs.Nx 2051 310
deg_Kelly.Hx.vs.Nx 5301 866
2A Nx
deg_Nx.Hif1a.vs.Kelly 223 21
deg_Nx.Hif2a.vs.Kelly 596 75
deg_Nx.Hif1b.vs.Kelly 721 58
2B Hx
deg_Hx.Hif1a.vs.Kelly 1013 77
deg_Hx.Hif2a.vs.Kelly 2532 348
deg_Hx.Hif1b.vs.Kelly 4208 531
deg_Hx.Hif2a.vs.Hif1a 4174 607
deg_Hx.Hif1b.vs.Hif1a 4972 643
deg_Hx.Hif1b.vs.Hif2a 2043 249
3 interaction
deg_Hif1aHxNx.vs.KellyHxNx 686 54
deg_Hif2aHxNx.vs.KellyHxNx 2238 281
deg_Hif1bHxNx.vs.KellyHxNx 3887 431
group contrasts
deg_Hx.Hif1b.vs.Hif12a 2741 224
deg_Hx.Kelly.vs.allHIFs 1502 148
deg_Hx.vs.Nx 3599 495

Which results to use?

Example for Hif1a

Results 1

SCARB1 doesn’t fit!

Kelly.Hx.vs.Nx

baseMean log2FoldChange lfcSE stat pvalue padj symbol
ENSG00000073060 11958.596 1.912261 0.0564790 33.85793 0 0 SCARB1
ENSG00000132382 6908.702 -1.840556 0.0836373 -22.00641 0 0 MYBBP1A
ENSG00000186469 8085.827 1.871876 0.0904267 20.70048 0 0 GNG2

Hif1a.Hx.vs.Nx

baseMean log2FoldChange lfcSE stat pvalue padj symbol
ENSG00000073060 11958.596 2.513715 0.0674164 37.28643 0 0 SCARB1
ENSG00000132382 6908.702 -2.577358 0.1000207 -25.76825 0 0 MYBBP1A
ENSG00000186469 8085.827 2.579589 0.1081358 23.85510 0 0 GNG2

compare results with contrast vsvs (Hif1a Hx vs. Nx VS. Kelly Hx vs. Nx